The overall goal of this application is to understand the molecular interactions between Nef and the cellular proteins essential for the role of Nef in AIDS pathogenesis. Molecular-genetic and biochemical approaches will be used to dissect several functions of HIV-1 and SIV Nef, including the ability of Nef to induce endocytosis of CD4 and MHC class I molecules, to alter cellular signal transduction pathways, and to enhance viral replication. These experiments will uncover multiple molecular interactions between Nef and components of the cellular signal transduction and endocytic machinery that underlie these functions. The importance of these molecular interactions of Nef for AIDS pathogenesis will be addressed using the SIV-infected rhesus monkey model and in studies of natural Nef proteins isolated directly from HIV-1 infected people. Specifically, the first two sets of experiments will characterize the interaction between Nef and the cytoplasmic domains of CD4 and MHC class I molecules using genetic and biochemical techniques and will identify mutations in Nef that dissociate individual functions and/or molecular interactions between Nef and the components of endocytic and signal transduction machineries. The third set of experiments will use the yeast two-hybrid system to identify cellular proteins that mediate functional interactions of Nef with the endocytic and signal transduction machineries. Finally, they will study the effect of mutations disrupting selected molecular interactions of Nef on AIDS pathogenesis in rhesus monkeys. These studies will provide a molecular framework for understanding the functions of Nef, the molecular interactions that underlie these functions and their importance for the pathogenic process. Results from their studies will have implications for a rational design of effective strategies for pharmacological disruption of Nef functions that are required for AIDS pathogenesis.